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Owing to the quality control mechanisms operating in the early secretory compartment, only native proteins are secreted. Despite the difficulties in assembling planar immunoglobulin M (IgM) polymers, antibody‐secreting cells can release up to thousands of IgM per second. The finding that secretory μ (μs) chains bind to ERGIC‐53, a lectin transporter that cycles in the early secretory compartment,...
Cubam is a multi‐ligand receptor involved in dietary uptake of intrinsic factor‐vitamin B12 in the small intestine and reabsorption of various low‐molecular‐weight proteins (such as albumin, transferrin, apolipoprotein A‐I and vitamin D‐binding protein) in the kidney. Cubam is composed of two proteins: cubilin and amnionless. Cubilin harbors ligand binding capabilities, while amnionless provides membrane anchorage and potential endocytic capacity via two FXNPXF signals within the cytosolic domain. These signals are similar to the FXNPXY signals found in members of the low‐density lipoprotein receptor superfamily, which associate with clathrin‐associated sorting proteins, including Disabled‐2 (Dab2) and autosomal recessive hypercholesterolemia (ARH), during endocytosis. We therefore investigated the functionality of each amnionless FXNPXF signal and their respective interaction with sorting proteins. By sequential mutation and expression of a panel of amnionless mutants combined with yeast two‐hybrid analyses, we demonstrate that the signals are functionally redundant and both are able to mediate endocytosis of cubam through interaction with Dab2 and ARH....
Exosomes play important roles in many physiological and pathological processes. However, the exosome–cell interaction mode and the intracellular trafficking pathway of exosomes in their recipient cells remain unclear. Here, we report that exosomes derived from K562 or MT4 cells are internalized more efficiently by phagocytes than by non‐phagocytic cells. Most exosomes were observed attached to the plasma membrane of non‐phagocytic cells, while in phagocytic cells these exosomes were found to enter via phagocytosis. Specifically, they moved to phagosomes together with phagocytic polystyrene carboxylate‐modified latex beads (biospheres) and were further sorted into phagolysosomes. Moreover, exosome internalization was dependent on the actin cytoskeleton and phosphatidylinositol 3‐kinase, and could be inhibited by the knockdown of dynamin2 or overexpression of a dominant‐negative form of dynamin2. Further, antibody pretreatment assays demonstrated that tim4 but not tim1 was involved in exosomes uptake. We also found that exosomes did not enter the internalization pathway involving caveolae, macropinocytosis and clathrin‐coated vesicles. Our observation that the cellular uptake of exosomes occurs through phagocytosis has important implications for exosome–cell interactions and the exosome intracellular trafficking pathway....
We used multiple approaches to investigate the coordination of trans and medial Rab proteins in the regulation of intra‐Golgi retrograde trafficking. We reasoned that medially located Rab33b might act downstream of the trans Golgi Rab, Rab6, in regulating intra‐Golgi retrograde trafficking. We found that knockdown of Rab33b, like Rab6, suppressed conserved oligomeric Golgi (COG) complex‐ or Zeste...
Biogenesis of a specialized organelle that supports intracellular replication of Legionella pneumophila involves the fusion of secretory vesicles exiting the endoplasmic reticulum (ER) with phagosomes containing this bacterial pathogen. Here, we investigated host plasma membrane SNARE proteins to determine whether they play a role in trafficking of vacuoles containing L. pneumophila. Depletion of...
Membrane fusion is dependent on the function of SNAREs and their α‐helical SNARE motifs that form SNARE complexes. The Habc domains at the N‐termini of some SNAREs can interact with their associated SNARE motif, Sec1/Munc18 (SM) proteins, tethering proteins or adaptor proteins, suggesting that they play an important regulatory function. We screened for proteins that interact with the Habc domain of Syntaxin 6, and isolated an uncharacterized 164‐kDa protein that we named SHIP164. SHIP164 is part of a large (∼700 kDa) complex, and interacts with components of the Golgi‐associated retrograde protein (GARP) tethering complex. Depletion of GARP subunits or overexpression of Syntaxin 6 results in a redistribution of soluble SHIP164 to endosomal structures. Co‐overexpression of Syntaxin 6 and SHIP164 produced excessive tubulation of endosomes, and perturbed the transport of cation‐independent mannose‐6‐phosphate receptor (CI‐MPR) and transferrin receptor. Thus, we propose that SHIP164 functions in trafficking through the early/recycling endosomal system....
Biogenesis of lysosome‐related organelles complex (BLOC)‐1, ‐2 and ‐3 are three multi‐subunit protein complexes that are deficient in various forms of Hermansky‐Pudlak syndrome, a human disease characterized by abnormal formation of lysosome‐related organelles. Contrasting views have arisen on the evolutionary origin of these protein complexes. One view is that the BLOCs represent a recent evolutionary...
Human cytomegalovirus (HCMV) encodes the seven transmembrane (7TM)/G‐protein coupled receptor (GPCR) US28, which signals and endocytoses in a constitutive, ligand‐independent manner. Here we show that, following endocytosis, US28 is targeted to the lysosomes for degradation as a consequence of its interaction with the GPCR‐associated sorting protein‐1 (GASP‐1). We find that GASP‐1 binds to US28 in vitro...
Hormone‐ and neuropeptide‐containing secretory granules (SGs) of neuroendocrine PC12 cells are formed at the trans‐ Golgi network as immature SGs. These intermediates are converted to mature SGs in a complex maturation process, including matrix condensation, processing of cargo proteins and removal of proteins and membrane in clathrin‐coated vesicles. The resulting mature SGs undergo Ca2+‐dependent exocytosis upon an appropriate stimulus. We here show that the motor protein myosin Va is implicated in a maturation step of SGs, their binding to F‐actin and their stimulated exocytosis. Interference with myosin Va function blocked the removal of the transmembrane protein furin from maturing SGs without affecting condensation and processing of proteins of the SG lumen. Furthermore, the ATP‐inhibited binding of SGs to F‐actin decreased with progressive maturation and upon interference with myosin Va function. Moreover, the expression of a dominant‐negative myosin Va‐tail or shRNA‐based downregulation of myosin Va interfered with stimulated exocytosis of SGs. In summary, our data suggest an essential function of myosin Va in the membrane remodeling of SGs during maturation and a role in their exocytosis....
Little is known about the formation and regulation of endoplasmic reticulum (ER)–Golgi transport intermediates, although previous studies suggest that cargo is the main regulator of their morphology. In this study, we analyze the role of coat protein I (COPI) and cytoskeleton in the formation of tubular ER–Golgi intermediate compartment (ERGIC) and also show that partial COPI detachment by means of low temperature (15°C) or brefeldin A induces the formation of transient tubular ERGIC elements. Most of them moved from the cell periphery to the perinuclear area and were 2.5× slower than vesicles. Time‐lapse analysis of living cells demonstrates that the ERGIC elements are able to shift very fast from tubular to vesicular forms and vice versa, suggesting that the amount of cargo is not the determining factor for ERGIC morphology. Both the partial microtubule depolymerization and the inhibition of uncoating of the membranes result in the formation of long tubules that grow from round ERGICs and form at complex network. Interestingly, both COPI detachment and microtubule depolymerization induce a redistribution of kinesin from peripheral ERGIC elements to the Golgi area, while dynein distribution is not affected. However, both kinesin and dynein downregulation by RNA interference induced ERGIC tubulation. The tubules induced by kinesin depletion were static, whereas those resulting from dynein depletion were highly mobile. Our results strongly suggest that the interaction of motor proteins with COPI‐coated membranes and microtubules is a key regulator of ERGIC morphology and mobility....
Different primary lysosomal trafficking defects lead to common alterations in lipid trafficking, suggesting cooperative interactions among lysosomal lipids. However, cellular analysis of the functional consequences of this phenomenon is lacking. As a test case, we studied cells with defective Niemann‐Pick C1 (NPC1) protein, a cholesterol trafficking protein whose defect gives rise to lysosomal accumulation...
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